Clonal analysis and dynamic imaging identify multipotency of individual Gallus gallus caudal hindbrain neural crest cells toward cardiac and enteric fates

Weiyi Tang,Yuwei Li,Marianne E. Bronner,Ang Li

doi:10.1038/s41467-021-22146-8

Weiyi Tang, Yuwei Li + Show 2 more

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https://doi.org/10.1038/s41467-021-22146-8

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Abstract

Neural crest stem cells arising from caudal hindbrain (often called cardiac and posterior vagal neural crest) migrate long distances to form cell types as diverse as heart muscle and enteric ganglia, abnormalities of which lead to common congenital birth defects. Here, we explore whether individual caudal hindbrain neural crest precursors are multipotent or predetermined toward these particular fates and destinations. To this end, we perform lineage tracing of chick neural crest cells at single-cell resolution using two complementary approaches: retrovirally mediated multiplex clonal analysis and single-cell photoconversion. Both methods show that the majority of these neural crest precursors are multipotent with many clones producing mesenchymal as well as neuronal derivatives. Time-lapse imaging demonstrates that sister cells can migrate in distinct directions, suggesting stochasticity in choice of migration path. Perturbation experiments further identify guidance cues acting on cells in the pharyngeal junction that can influence this choice; loss of CXCR4 signaling results in failure to migrate to the heart but no influence on migration toward the foregut, whereas loss of RET signaling does the opposite. Taken together, the results suggest that environmental influences rather than intrinsic information govern cell fate choice of multipotent caudal hindbrain neural crest cells.

Highlights

Neural crest stem cells arising from caudal hindbrain migrate long distances to form cell types as diverse as heart muscle and enteric ganglia, abnormalities of which lead to common congenital birth defects
A slice was cut directly posterior to the otic placode (Fig. 1b) to obtain a transverse view of all cardiac neural crest-related structures (Fig. 1c, gray), including the dorsal neural tube, cranial nerve nine (CN-IX), pharyngeal arch arteries (PAA), and outflow tract (OFT)
Staining with the neuronal marker HuC/D at embryonic day 7 (E7), a time after neuronal differentiation, identified one cell at the center of CN-IX that expressed the neuronal marker HuC/D (Fig. 1j), but another HuC/D negative cell of the same clone at the periphery of the ganglion, a location where presumptive glial cells often reside, consistent with a presumptive non-neuronal cell fate (Fig. 1k). These results suggest that individual premigratory cardiac neural crest cells can produce diverse cell types even when in close proximity, indicating that location alone may underestimate the developmental potential within a clone

Summary

Introduction

Neural crest stem cells arising from caudal hindbrain (often called cardiac and posterior vagal neural crest) migrate long distances to form cell types as diverse as heart muscle and enteric ganglia, abnormalities of which lead to common congenital birth defects. The question of multipotency is pertinent to neural crest cells arising from the caudal hindbrain, which undergo some of the longest migrations of any embryonic cell type and populate derivatives as diverse as muscle of the heart[3,4], and neurons and glia of the sympathetic and enteric nervous system (ENS)[5]. These population level analyses clearly show that cells delaminating from the cardiac level can contribute to both cardiovascular system and the ENS, albeit the source of the cells, whether of neural crest and/or Schwann cell precursor origin, remains a matter of debate[8,9] These studies raise the intriguing question of whether individual neural crest cells are multipotent, with the potential to form both cardiac and enteric derivatives, or if there are distinct precursors fated to populate each of these distant destinations that migrate differentially. While these experiments have tested the potential of cardiac neural crest cells removed from the embryonic milieu and grown at clonal density, it is technically difficult to extend these analyses in vivo

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